, 2002, Koch et al., 2008, Massaro et al., 2009, Pielage et al., 2005 and Pielage et al., 2008). Staining hts mutant animals with additional pre- and postsynaptic markers further supports this conclusion (see Figure S1B available online). The frequency and severity

of synapse retractions were quantified in wild-type and hts mutant animals. Wild-type animals show virtually no evidence of synapse retraction, and when it does occur the retractions only encompass one or two synaptic boutons ( Figures 2E and 2F; WT retraction 3.3%, n = 120). By contrast, hts mutations have a large increase in both the frequency and severity of NMJ retractions (23%–53%, n > 100) both on muscle 4 and muscles

6/7 (Figures 2E, 2F, S8). In addition, the frequency and severity of this phenotype correlates Dolutegravir well with the molecular nature of Panobinostat solubility dmso our mutant alleles with hts1103/DfBSC26 representing a strong hypomorph or null combination and showing the most severe phenotype. It is worth noting that the htsΔG mutation, which results in a truncation before the C-terminal MARCKS domain, shows a significant increase in NMJ retractions compared to wild-type, suggesting the importance of the Hts/Adducin actin-binding and capping activity for synapse stability ( Figures 2E and 2F). However, the phenotype of the htsΔG mutation is not as severe Ampicillin as the null mutation. One possibility is that the truncated protein retains some actin-capping activity as indicated by in vitro studies ( Li et al., 1998). Alternatively, there remains a

stabilizing function that is independent of the MARCKS domain in vivo that is not predicted from the in vitro data. From these data, we conclude that Hts-M is required for the stabilization of the presynaptic nerve terminal. Adducin interacts with the Spectrin skeleton in Drosophila and other systems ( Bennett and Baines, 2001). The demonstration that Hts/Adducin is necessary for synapse stability is consistent with prior studies demonstrating that presynaptic α-/β-Spectrin and the spectrin-interacting adaptor protein Ankyrin2 are required for synapse stability ( Koch et al., 2008, Pielage et al., 2005 and Pielage et al., 2008). Indeed, the severity of synapse retraction and elimination is comparable in all three mutant genotypes. Consistent with this correlation, we observe a loss of the cell adhesion molecule Fasciclin II, the microtubule associated protein Futsch and Ankyrin2L (Ank2L) within retracting portions of the NMJ ( Figures S1C–S1H). In addition, we observe that Ank2L staining is perturbed within stable regions of the NMJ ( Figure S1H). Finally, loss of Hts/Adducin potentially has a minor impact on axonal transport. In hts mutant animals, we observe increased levels of synaptic antigens in the axon.